Stem Cells
The embryonal stem (ES) cell has unlimited self-renewal and can differentiate into all tissue types. ES cells are derived from the inner cell mass of the blastocyst or primordial germ cells from a post-implantation embryo (embryonal germ cells or EG cells). ES and EG cells have been derived from mouse, and, more recently, from non-human primates and humans. When introduced into blastocysts, ES cells can contribute to all tissues. A drawback to ES cell therapy is that, when transplanted in post-natal animals, ES and EG cells generate teratomas.
ES (and EG) cells can be identified by positive staining with antibodies to SSEA1 (mouse) and SSEA4 (human). At the molecular level, ES and EG cells express a number of transcription factors specific for these undifferentiated cells. These include Oct-4 and rex-1. Also found are the LIF-R (in mouse) and the transcription factors sox-2 and rox-1. Rox-1 and sox-2 are also expressed in non-ES cells. A hallmark of ES cells is the presence of telomerase, which provides these cells with an unlimited self-renewal potential in vitro.
Oct-4 (Oct 3 in humans) is a transcription factor expressed in the pregastrulation embryo, early cleavage stage embryo, cells of the inner cell mass of the blastocyst, and in embryonic carcinoma (EC) cells (Nichols J, et al (1998) Cell 95:379-91), and is down-regulated when cells are induced to differentiate. Expression of Oct-4 plays an important role in determining early steps in embryogenesis and differentiation. Oct-4, in combination with Rox-1, causes transcriptional activation of the Zn-finger protein Rex-1, also required for maintaining ES undiffereniated (Rosfjord E, Rizzino A. (1997) Biochem Biophys Res Commun 203:1795-802; Ben-Shushan E, et al (1998) Mol Cell Biol 18:1866-78). In addition, sox-2, expressed in ES/EC, but also in other more differentiated cells, is needed together with Oct-4 to retain the undifferentiated state of ES/EC (Uwanogho D et al (1995) Mech Dev 49:23-36). Maintenance of murine ES cells and primordial germ cells requires LIF.
The Oct 4 gene (Oct 3 in humans) is transcribed into at least two splice variants in humans, Oct 3A and Oct 3B. The Oct 3B splice variant is found in many differentiated cells whereas the Oct 3A splice variant (also previously designated Oct ¾) is reported to be specific for the undifferentiated embryonic stem cell. See Shimozaki et al (2003) Development 130:2505-12.
Adult stem cells have been identified in most tissues. Hematopoietic stem cells are mesoderm-derived and have been purified using cell surface markers and functional characteristics. The hematopoietic stem cell, isolated from bone marrow, blood, cord blood, fetal liver and yolk sac, is the progenitor cell that reinitiates hematopoiesis and generates multiple hematopoietic lineages. Hematopoietic stem cells can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell pool. Stem cells that differentiate only to form cells of hematopoietic lineage, however, are unable to provide a source of cells for repair of other damaged tissues, for example, heart.
Neural stem cells were initially identified in the subventricular zone and the olfactory bulb of fetal brain. Studies in rodents, non-human primates and humans, have shown that stem cells continue to be present in adult brain. These stem cells can proliferate in vivo and continuously regenerate at least some neuronal cells in vivo. When cultured ex vivo, neural stem cells can be induced to proliferate and differentiate into different types of neurons and glial cells. When transplanted into the brain, neural stem cells can engraft and generate neural cells and glial cells.
Mesenchymal stem cells (MSC), originally derived from the embryonal mesoderm and isolated from adult bone marrow, can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Mesoderm also differentiates into visceral mesoderm which can give rise to cardiac muscle, smooth muscle, or blood islands consisting of endothelium and hematopoietic progenitor cells. All of the many mesenchymal stem cells that have been described have demonstrated limited differentiation to cells generally considered to be of mesenchymal origin. To date, the best characterized mesenchymal stem cell reported is the cell isolated by Pittenger, et al. (Science (1999) 284: 143-147 and U.S. Pat. No. 5,827,740 (SH2+ SH4+ CD29+ CD44+ CD71+ CD90+ CD106+ CD120a+ CD124+ CD14−CD34− CD45−)). This cell is apparently limited in differentiation potential to cells of the mesenchymal lineage.
There is a need, therefore, for a non-embryonic stem cell that has the capacity to form differentiated cells of more than one embryonic lineage.
Myocardial Infarct (MI)
Myocardial infarction (MI) is characterized by the death of myocytes, coagulative necrosis, myocytolysis, contraction band necrosis, or apoptosis, resulting from a critical imbalance between the oxygen supply and demand of the myocardium. The most common cause of MI is coronary artery thrombosis following the rupture of atheromatous plaques. Though once strictly defined as a lack of blood flow, the modern definition of ischemia emphasizes the imbalance between oxygen supply and demand as well as the inadequate removal of metabolic waste products. Impaired oxygen delivery results in a reduction in oxidative phosphorylation that causes anaerobic glycolysis. This produces excess lactate that accumulates in the myocardium. Impaired ATP production and acidosis results in a decline in myocardial contractility. Similarly, ischemia reperfusion injury, without total occlusion, can also cause cardiac damage The exposure of the contents of the plaque to the basement membrane following plaque rupture ultimately results in vessel blockage culminating from a series of events including platelet aggregation, thrombus formation, fibrin accumulation, and vasospasm. Total occlusion of the vessel for more than 4-6 hours results in irreversible myocardial necrosis. Ultimately, death and morbidity from myocardial infarction is the result of fatal dysrhythmia or progressive heart failure. Progressive heart failure is chiefly the result of insufficient muscle mass (deficiency in muscle cells) or improper function of the heart muscle, which can be caused by various conditions including, but not limited to, hypertension. Progressive heart failure is, therefore, the focus of cell-based therapy.
All current strategies for the treatment of myocardial infarction focus on limiting myocyte death. Annually in the United States, 500,000 patients undergo angioplasty with stent placement. 400,000 will undergo coronary artery bypass, while an unknown additional number of patients will be treated by thrombolytic therapy.
One approach, known as cellular cardiomyoplasty, has received recent attention and focuses on repopulation and engraftment of the injured myocardium by transplantation of healthy cells (Reffelmann, T. and Kloner, R. A. (2003) Cardiovasc Res. 58 (2): 358-68). Many cell types that might replace necrotic tissue and minimize regional scarring have been considered. Cells that have already committed to a specific lineage, such as satellite cells, cardiomyocytes, primary myocardial cell cultures, fibroblasts, and skeletal myoblasts, have been readily used in cellular cardiomyoplasty with limited success in restoring damaged tissue and improving cardiac function (Menasche, P. (2003) Cardiovasc Res. 58 (2): 351-7; Etzion, S. et al (2001) J Mol Cell Cardiol. 33 (7): 1321-30; Sakai, T. et al (1999) J. Thorac. Cardiovasc. Surg. 118 (4): 715-24).
Cardiogenic progenitors are precursor cells that have committed to the cardiac lineage, but have not differentiated into cardiac muscle. Cardiomyocytes are the cells that comprise the heart. They are also known as cardiac muscle cells. Use of cardiomyocytes in the repair of cardiac tissue has been proposed. However, this approach is hindered by an inability to obtain sufficient quantities of cardiomyocytes for the repair of large areas of infarcted myocardium. Doubt has also been cast over the incorporation and tissue-specific function of intra-cardiac grafts derived from cardiomyocytes, even when they are harvested from embryonic sources (Etzion, S. et al (2001) J. Mol. Cell. Cardiol. 33 (7): 1321-30). Intra-cardiac grafts using this cell type can be successfully grafted and are able to survive in the myocardium after permanent coronary artery occlusion and extensive infarction. However, engrafted rat embryonic cardiomyocytes attenuate, but do not fully reverse, left ventricular dilatation and prevent wall thinning. While survival was improved during 8 weeks of follow-up, the implanted cells did not develop into fully differentiated myocardium. Surprisingly, they remained isolated from the host myocardium by scar tissue and did not improve systolic function over time (Etzion, S. et al (2001) J. Mol. Cell. Cardiol. 33 (7): 1321-30).
Congestive Heart Failure
Congestive heart failure (CHF) is a clinical condition in which a primary or secondary circulatory system disease causes abnormal cardiac pressure or performance characteristics that lead to pulmonary congestion (Zhang, J. and Narula, J. (2004) Surg Clin N Am 84:223-242). CHF is a chronic condition that results when the heart muscle is unable to pump blood as efficiently as is needed to maintain physiological homeostasis. Although common causes of CHF include hypertension, anemia and cardiomyopathy, CHF is most often caused by myocardial infarction (Lee, Michael, et al (2004) Reviews in Cardiovascular Medicine, 5: 82-94). Currently, nearly 550,000 new cases of heart failure are now diagnosed each year.
CHF occurs when cardiac dysfunction prevents adequate perfusion of peripheral tissues. Inadequate perfusion leads to stimulation of compensatory mechanisms that then cause many of the clinical signs and symptoms of the condition. In patients with CHF, neurohumoral compensatory mechanisms are activated. Chronic stimulation by these agents increases cardiac afterload and preload, further worsening ventricular function.
Small molecule therapeutics are being tested with the aim to disrupt these compensatory systems. However, no pharmacological intervention improves the underlying pathophysiology of CHF.
Surgical intervention to treat CHF is also limited. Cardiac transplantation is the mainstay of treatment for patients with end-stage cardiomyopathies, such as CHF, but is limited by the scarcity of donor organs and complications, such as graft rejection and allograft coronary vasculopathy (Fedak, P. et al (2003) Seminars in Thoracic and Cardiovascular Surgery, 15: 277-286).
Recent studies challenge the traditional dogma that the heart is a terminally differentiated post-mitotic organ incapable of self-renewal. This opens the door to the possibility of delivering cells to treat CHF. It would be desirable to repopulate the injured cells with stem cells to regenerate cardiomyocytes and blood vessels, reverse ventricular remodeling, or reduce apoptosis of existing cells. Both myogenesis and angiogenesis may be required to restore cardiac function in patients with transmural scar tissue. Thus, cells that are capable of inducing angiogenesis and forming muscle-like cells and endothelial cells may be useful to reverse cardiac dysfunction in patients with CHF.
Clinical Experience with Stem Cells to Treat Cardiovascular Disease
Given the feasibility of the procedure, autologous skeletal muscle cell transplantation has been used clinically (Menasche P et al (2001) Lancet 357:279-280). Autologous skeletal myoblasts were directly injected into nonviable regions of the heart at the time of coronary bypass grafting or LVAD insertion. They formed viable grafts (Pagani F et al (2002) Circulation 106:II463 (abstract)). Patients reported improved symptoms and global heart functions. There was also evidence of viability in the dead region after clinical cell transplantation.
Both autologous bone marrow cells and myoblasts have been used clinically in humans in a CHF setting. Strauer and co-workers transplanted autologous mononuclear bone marrow cells in patients with acute myocardial infarction (Trauer et al (2002) Circulation 106: 1913-1918). Regions of cell delivery demonstrated increased perfusion, viability, and wall motion, demonstrating improved myogenesis and angiogenesis.
Bone marrow cell-derived progenitor cells and circulating progenitor cells have also been tested (Assmus et al (2002) Circulation 106:3009-3017). Treatment was associated with improved regional and global heart function, improved viability in the infarct area, and reduced left ventricular end-systolic volumes.
Human ES cells have been demonstrated to differentiate into myocytes with many of the functional characteristics of cardiomyocytes (Cai, J. (2002) Neuromolecular Med,. 2 (3): 233-49). There are, however, safety concerns regarding therapeutic use because ES cells cause the formation of teratomas when administered to animals.
Adult stem cells that are dispersed throughout normal adults and can be isolated from a number of tissue sources, including organs, bone marrow and blood. They are free from many of the ethical and safety issues associated with ES cells (Cai, J. (2002) Neuromolecular Med,. 2 (3): 233-49) and have not been linked to the growth of teratomas or cancerous tumors. However, adult stem cells have shown limitations in their potential for therapeutic applications in that their differentiation potential generally appears to be restricted to narrowly-defined cell lineages or tissues, typically reflecting the tissue or organ from which the cells were isolated (Raff, M. (2003) Annu Rev Cell Dev Biol 19: 1-22; Verfaillie, C. M. (2002) Trends Cell Biol,. 12 (11): 502-8).
Hematopoietic stem cells (HSC) have been utilized therapeutically for several decades in immune reconstitution settings.
In summary, loss of function and/or cell mass in cardiac muscle can arise, for example, by physical damage or disease-related damage (e.g., genetic or acquired disease). Stem cell technology has made cellular myoplasty a realistic treatment for restoring or enhancing cardiac muscle function or cell mass. Tissue-specific stem cells and embryonic stem cells provide limited results.